SETD2 deficiency promotes renal fibrosis through the TGF‐β/Smad signalling pathway in the absence of VHL

Abstract Background Renal fibrosis is the final development pathway and the most common pathological manifestation of chronic kidney disease. Epigenetic alteration is a significant intrinsic factor contributing to the development of renal fibrosis. SET domain‐containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, catalysing H3K36 trimethylation. There is evidence that SETD2‐mediated epigenetic alterations are implicated in many diseases. However, it is unclear what role SETD2 plays in the development of renal fibrosis. Methods Kidney tissues from mice as well as HK2 cells were used as research subjects. Clinical databases of patients with renal fibrosis were analysed to investigate whether SETD2 expression is reduced in the occurrence of renal fibrosis. SETD2 and Von Hippel–Lindau (VHL) double‐knockout mice were used to further investigate the role of SETD2 in renal fibrosis. Renal tubular epithelial cells isolated from mice were used for RNA sequencing and chromatin immunoprecipitation sequencing to search for molecular signalling pathways and key molecules leading to renal fibrosis in mice. Molecular and cell biology experiments were conducted to analyse and validate the role of SETD2 in the development of renal fibrosis. Finally, rescue experiments were performed to determine the molecular mechanism of SETD2 deficiency in the development of renal fibrosis. Results SETD2 deficiency leads to severe renal fibrosis in VHL‐deficient mice. Mechanically, SETD2 maintains the transcriptional level of Smad7, a negative feedback factor of the transforming growth factor‐β (TGF‐β)/Smad signalling pathway, thereby preventing the activation of the TGF‐β/Smad signalling pathway. Deletion of SETD2 leads to reduced Smad7 expression, which results in activation of the TGF‐β/Smad signalling pathway and ultimately renal fibrosis in the absence of VHL. Conclusions Our findings reveal the role of SETD2‐mediated H3K36me3 of Smad7 in regulating the TGF‐β/Smad signalling pathway in renal fibrogenesis and provide an innovative insight into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.


INTRODUCTION
Chronic kidney disease (CKD) is a progressive disease with no cure and affects approximately 10% of adults worldwide. 1Renal fibrosis is the final development pathway and the most common pathological manifestation of CKD. 2 Characterised by deposition of extracellular matrix, such as collagen type I alpha 1 chain (COL1A1) and fibronectin (FN), renal fibrosis causes tissue scarring and ultimately leads to end-stage renal disease. 3For the majority of individuals suffering from kidney failure, kidney transplantation and dialysis remain the prevailing treatment options.5][6][7] So, it is crucial to identify and detect new potential targets to prevent renal fibrogenesis.
There is compelling literature suggesting increased expression and activation of transforming growth factor-β (TGF-β) in human kidney disease. 8,9In addition, numerous animal studies have been conducted to confirm that the TGF-β/Smad signalling pathway plays a pivotal role in driving the progression of renal fibrosis. 10,11TGF-β/Smad signalling pathway acts through a very typical signalling pathway that includes the phosphorylation and activation of Smad2 and Smad3 by the TGF-β/Smad signalling pathway receptor 1 (TGFR1, also known as ALK5).Subsequently, the phosphorylated Smad2 and Smad3 bind Smad4 to form a complex that translocates into the nucleus, thereby facilitating the transcription of target genes involved in renal fibrosis. 12This process is inhibited by the inhibitory Smad, including Smad6 and Smad7.As a negative feedback inhibitor of the TGF-β/Smad signalling pathway, Smad7 recruits E3 ubiquitin ligase SMADubiquitination-regulatory factor 1 (Smurf1) and Smurf2 to degrade TGFR1. 13In addition, Smad7 inhibits the TGFβ/Smad signalling pathway by competing with Smad2/3 for bounding to TGFR1 to prevent the phosphorylation of Smad2/3. 14 important intrinsic cause of CKD is epigenetic alteration. 15Epigenetics refers to changes in the transcription and expression of genes rather than changes in the genes themselves, including methylation of DNA, histone modification and non-coding RNA. 16There are compelling studies suggesting that histone modifications play a crucial role in CKD and renal fibrogenesis, such as H3K9me2/3, H3K4me1/2/3, and H3K27me3. 17,18However, the role of H3K36 methylation in renal fibrosis remains unknown.SET domain-containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, which catalyses H3K36 trimethylation. 19][28][29][30][31][32][33][34][35] Notably, SETD2 deficiency accelerates the transition from polycystic kidney disease (PKD) to RCC by regulating βcatenin activity. 26However, the role of SETD2 in renal fibrosis remains still unknown.
It is worth noting that Von Hippel-Lindau (VHL) is an important regulatory molecule in the pathogenesis of kidney diseases.][41] Here, we established a mouse model of renal fibrosis driven by the inactivation of SETD2 and VHL.We found that SETD2 can maintain the transcriptional level of Smad7 through H3K36me3, thus inhibiting the activation of the TGF-β/Smad signalling pathway.SETD2 loss results in decreased Smad7 level and TGF-β/Smad signalling pathway activation, which is predictive of fibrosis in the absence of VHL.

Mouse strains
Setd2 Male and female VHL −KO ; Setd2 −KO mice, as well as VHL −KO mice, were euthanised at 15 and 30 weeks of age for subsequent experiments.Additionally, male wild-type (WT) and Setd2 −KO mice were euthanised on days 7 and 14 after unilateral ureteral obstruction (UUO) at 8 weeks of age (n = 10).

Blood urea nitrogen and creatinine test
Blood was collected from 15-week-old VHL −KO ; Setd2 −KO and VHL −KO mice (average weight = 30 g, n = 6) using the tail vein blood collection method.The serum was analysed for Blood urea nitrogen (BUN) and creatinine concentrations by Wuhan Servicebio on a Beckman Coulter AU680 analyser.

Isolation of primary tubular epithelial cells
To increase the percentage of Cre recombinase-positive cells, we separated primary renal tubular epithelial cells (PTECs) from 10-week-old VHL −KO ; Setd2 −KO and VHL −KO mice (average weight = 25 g, n = 10) kidneys.Previously described procedures for isolating and identifying phenotypes were followed. 42

RNA isolation and reverse transcription real-time quantitative PCR
RNA was isolated from 10-week-old VHL −KO ; Setd2 −KO and VHL −KO mouse PTECs and HK2 cells using TRIzol reagent (Invitrogen) Reverse transcription real-time quan-titative PCR (RT-qPCR) was performed using Setd2 and target gene primers with the Prime Script RT reagent kit (TaKaRa).GAPDH was used to normalise the results.Data were analysed from three independent experiments and are shown as the mean ± standard error of mean.

RNA sequencing and analyses
RNA was extracted from 10-week-old VHL −KO ; Setd2 −KO and VHL −KO mice PTECs.For the construction of sequencing libraries, New England Biolabs' Next Ultra Directional RNA Library Prep Kit for Illumina (Ipswich) was utilised.Afterward, Illumina sequencing was performed with paired-ends 2 × 150 as the sequencing mode.Differential gene expression was analysed using the DESeq2 package.The statistically significant DE genes were obtained by a p-value threshold <.05 and fold change ≥1.5.All differentially expressed mRNAs were selected for Gene Ontology (GO) analysis.

Chromatin immunoprecipitation-qPCR assay
Chromatin immunoprecipitation (ChIP) assays were performed using the EZ ChIP kit (Millipore).The procedure was performed as described in the kit provided by the manufacturer.Briefly, isolated PTECs were fixed with 1% formaldehyde and broken up by sonication.Immunoprecipitation was then performed with histone H3 trimethylK36 (Abcam, ab9050).After washing and reverse cross-linking, the precipitated DNA was amplified with primers and quantified by qPCR.

Masson and Sirius Red staining
Kidney samples were obtained from 15-and 30-week-old VHL −KO ; Setd2 −KO and VHL −KO mice.Kidney sections were stained with Masson's trichrome (Solar bio, G1340) and Picrosirius red (G-CLONE, RS1220).The experiment was performed in strict accordance with the operating instructions.Measurement of the fibrotic area was quantified with ImageJ software (NIH, http://rsbweb.nih.gov/ij/).

The mRNA expression of SETD2 is positively correlated with VHL in kidney
To investigate whether SETD2 expression is altered in fibrotic kidneys, we analysed clinical data from Keenan Research Centre for Biomedical Science and found reduced expression of SETD2 in patients with renal fibrosis (Figure 1A).Considering that fibrosis is usually the result of chronic inflammation, we also analysed biopsy data from patients with nephritis and patients with renal fibrosis, and the data showed that SETD2 expression is also decreased in patients with renal fibrosis compared to patients with inflammation (Figure 1B).In addition, we generated a mouse model of UUO, commonly used in studies of renal fibrosis, [42][43][44] to evaluate the expression level of SETD2 in renal fibrosis.We conducted double IF experiments using antibodies against Lotus tetragonolobus lectin (LTL) and THP, which are markers of proximal and distal tubules, respectively, to identify the tubules with SETD2 expression.As shown in Figure 1C,D, SETD2 is mainly expressed in the renal tubular epithelial cells in WT mice.And the expression level of SETD2 protein was decreased in the renal tubular epithelial cells in the kidney with renal fibrosis, especially in distal tubules.Meanwhile, considering that VHL deficiency can result in increased inflammation and fibrosis, 45 we explored the relationship between SETD2 and VHL.The mRNA expression of VHL in the kidneys with renal fibrosis was also reduced compared to that in normal kidneys (Figure 1E).Meanwhile, the mRNA expression of SETD2 is positively correlated with VHL in both kidneys with renal fibrosis and normal kidneys (Figure 1F-H).

Loss of SETD2 is no more prone to promote renal fibrosis in the UUO model
To explore the potential function of SETD2 in renal fibrosis in vivo, we used a transgenic Ksp1.3/Cre mouse line to generate Setd2 fl/fl mice and delete SETD2 in tubular epithelial cells (referred to as Setd2 −KO mice). 42First, we generated the UUO model of Setd2 −KO mice and WT mice (Figure 2A,B).Using Masson's trichrome and Sirius Red staining, we observed that the loss of SETD2 did not significantly increase the positive area of Masson's trichrome and Sirius Red staining compared to WT mice at either day 7 or 14 post-UUO.In addition, the IHC staining results also showed that the expression levels of α-SMA and COL1A1 remained unchanged in Setd2 −KO mice compared to WT mice (Figure 2C).These results indicated that Setd2 −KO mice were no more prone to develop renal fibrosis compared to WT mice under the condition of UUO.

SETD2 deficiency resulted in severe renal fibrosis in VHL-deficient mice
To further investigate whether SETD2 contributes to renal fibrosis, we generated Ksp Cre ; VHL fl/fl mice (hereafter referred to as VHL −KO mice) and Ksp Cre ; VHL fl/fl ; Setd2 fl/fl mice (hereafter referred to as VHL −KO ; Setd2 −KO mice)  3A).Based on the experimental results obtained from Western blotting (WB), IHC and RT-qPCR, we observed a downregulation of VHL, SETD2 and H3K36me3 expression levels in the renal tubules (Figure 3B-D).Observing the mouse kidneys, we found that the kidneys appeared to be crumpled and smaller in VHL −KO ; Setd2 −KO mice than in VHL −KO mice (Figure 3E).To observe the morphologic alteration, we performed H&E, Masson's trichrome and Sirius Red staining, and the results showed that VHL −KO ; Setd2 −KO mice exhibited a loss of normal renal tubular structure and numerous cysts.Additionally, these mice displayed characteristic features of renal fibro-sis (Figure 3F,G).Furthermore, we performed RT-qPCR and found a significant upregulation in the expression of inflammatory cytokines and chemokines in VHL −KO ; Setd2 −KO mice compared with VHL −KO mice, including Interleukin-1α (IL-1α), Interleukin-6 (IL-6), Chemokine (C-X-C motif) ligand 1 (CXCL1) and Chemokine (C-C motif) ligand2 (CCL2).Similarly, IF staining revealed an increased infiltration of macrophages and CD8 + T cells in VHL −KO ; Setd2 −KO mice compared with VHL −KO mice (Figure 3H,I).Meanwhile, the expression levels of COL1A1, α-SMA and FN in VHL −KO ; Setd2 −KO mice were much higher than that in VHL −KO mice, suggesting that 4 The inactivation of SET domain-containing 2 (SETD2) caused hyperactive transforming growth factor-β (TGF-β)/Smad signalling pathway in Von Hippel-Lindau (VHL)-deficient renal tubular epithelial cells.(A) Heatmap of genes with significantly different expression in freshly isolated renal tubular epithelial cells based on unsupervised hierarchical agglomerative clustering (n = 3 mice per SETD2 deficiency leads to renal fibrosis (Figure 3J,K).Moreover, in line with the morphologic alteration, BUN and creatinine levels were reduced in the blood of VHL −KO ; Setd2 −KO mice compared to the control group (Figure 3L).Together, these results indicated that SETD2 deficiency resulted in severe renal fibrosis in VHL-deficient mice.

The inactivation of SETD2 caused hyperactive TGF-β/Smad signalling pathway in VHL-deficient renal tubular epithelial cells
To explore the mechanisms by which SETD2 deficiency promotes renal fibrosis in VHL-deficient renal tubular epithelial cells, we performed RNA sequencing using PTECs freshly isolated from 10-week-old VHL −KO and VHL −KO ; Setd2 −KO mice.The overall transcriptome of VHL −KO ; Setd2 −KO PTECs was significantly altered compared to VHL −KO PTECs.A total of 2659 genes were upregulated and 2617 genes downregulated with a fold change ≥1.5 (Figure 4A).GO and pathway analysis presented that SETD2 inactivation significantly enriched lots of genes associated with pathways associated with fibrogenesis.In addition, GO analysis showed that several pathways were upregulated in VHL −KO ; Setd2 −KO mice compared with VHL −KO mice, including the TGF-β/Smad signalling pathway and PI3K-Akt signalling pathway (Figure 4B).We conducted gene set enrichment analysis to gain a better understanding of the signalling pathways mediated by SETD2.The data showed that TGF-β/Smad signalling pathway-associated genes were enriched in VHL −KO ; Setd2 −KO PTECs.To verify the results of the pathway analysis and explore the pivotal pathway in fibrogenesis, we used RT-qPCR to detect the expression levels of related pathways in more samples, including the TGFβ/Smad signalling pathway, PI3K-Akt signalling pathway, FoxO signalling pathway and Wnt signalling pathway.And the results showed that only the TGF-β/Smad signalling pathway was indeed significantly activated in VHL −KO ; Setd2 −KO mice (Figure 4D).TGF-β/Smad signalling pathway plays an important role in renal fibrogenesis and a wide range of studies have established the TGF-β/Smad signalling pathway as the paramount pathogenic factor that drives fibrosis.The activated Smad2 and Smad3 have been verified in VHL −KO ; Setd2 −KO mice by WB (Figure 4E).Furthermore, the data of IHC also showed that the positive rate of p-Smad3 was much higher in VHL −KO ; Setd2 −KO mice than that in VHL −KO mice (Figure 4F).These results indicated that SETD2 can inhibit the TGFβ/Smad signalling pathway transcriptional activity and SETD2 loss leads to p-Smad2 and p-Smad3 activation, which activates TGF-β/Smad signalling in VHL-deficient kidneys.

SETD2 inhibits TGF-β/Smad signalling activation by regulating Smad7 expression
To explore the potential mechanisms of SETD2-dependent epigenetic alterations and to identify genes associated with SETD2 and H3K36me3 on a genome-wide scale in the kidney, we referred to the ChIP sequencing data as described to explore possible SETD2/H3K36me3 targets. 26Considering that SETD2 can facilitate gene transcription, we investigated the negative regulators of the TGF-β/Smad signalling pathway and we found that the H3K36me3 antibody occupies the Smad7 transcription start region and exon region (Figure 5A).To demonstrate the involvement of SETD2 and H3K36me3 histone marker in Smad7 transcription, we verified the occupancy of H3K36me3 in the Smad7 transcribed region using ChIP-qPCR (Figure 5B).These results indicated that SETD2 deletion eliminates H3K36me3 modification in the transcriptional region Smad7.In addition, Smad7 protein expression was also reduced in VHL −KO ; Setd2 −KO mice compared with VHL −KO mice (Figure 5C).Furthermore, IHC and RT-qPCR results also showed that SETD2 loss resulted in the downregulation of Smad7 expression in VHL −KO ; Setd2 −KO mice PTECs (Figure 5D,E).To better verify the fact that SETD2 can regulate Smad7 transcription in vitro, we utilised renal cell lines and demonstrated that SETD2 depletion leads to a reduction in Smad7 transcript level, while overexpression of SETD2 leads to a large increase in Smad7 expression (Figure 5F).In addition, clinical data also showed a positive correlation between SETD2 and Smad7 expression (Figure 5G).Together, these results showed that SETD2 inhibits TGF-β/Smad signalling activation by regulating Smad7 expression.Notably, RT-qPCR analysis showed that group).(B) Pathway analysis of differentially expressed genes belonging to signalling pathways associated with SETD2 deletion.(C) Gene set enrichment analysis (GSEA) enrichment plots of differentially expressed genes associated with SETD2 deletion in VHL-deficient tissue.VHL mRNA expression level was reduced by SETD2 deficiency in the PETCs from 10-week-old mice (Figure 5H).This result suggested that SETD2 loss also partially affects the inhibitory function of VHL on the TGF-β signalling pathway.Furthermore, in VHL −KO ; Setd2 −KO mice PTECs, VHL is deleted in the renal tubules, leading to activation of the TGF-β/Smad signalling pathway.

SETD2 deletion leads to fibrosis through activation of the TGF-β/Smad signalling pathway in vitro
To further validate the effect of SETD2 on the TGFβ/Smad signalling pathway and exclude the effect of the pro-fibrotic environment, we deleted SETD2 through the CRISPR-Cas9 method in HK2 cells.SETD2 deletion resulted in increased expression of p-Smad3 and decreased expression of Smad7 in HK2 (Figure 6A,B).IF results presented that SETD2 deficiency resulted in the activation of the TGF-β/Smad signalling pathway (Figure 6C,D).In addition, the mRNA levels of COL1A1, COL1A2 and FN1, which are the target genes of p-Smad2/3, are increased in the absence of SETD2 (Figure 6E).IF results showed that FN protein expression levels increased after SETD2 deletion (Figure 6F).Considering that SETD2 inhibits TGF-β/Smad signalling activation by regulating Smad7 expression, we performed a gene rescue experiment with Smad7 overexpression in HK2 cells.And the results showed that Smad7 overexpression inhibited the TGFβ/Smad signal pathway caused by SETD2 and VHL deletion (Figure 6G).In addition, we further confirmed that SETD2 overexpression could facilitate Smad7 transcription to inhibit the TGF-β/Smad signalling pathway and renal fibrosis (Figure 6H).We also found that TGF-β can repress SETD2 expression (Figure 6I).These results indicated that SETD2 deletion leads to fibrogenesis through activation of the TGF-β/Smad signalling pathway in vitro.

Treatment with TGF-β/Smad inhibitor rescues the renal fibrosis phenotype caused by SETD2 absence
Given that SETD2 loss-mediated fibrogenesis is dependent on the hyperactivation of TGF-β/Smad signalling, we next investigated whether this phenotype could be rescued efficiently by the TGF-β/Smad signalling inhibitor.SIS3 is a novel specific Smad3 inhibitor that inhibits TGF-β by inhibiting Smad3 phosphorylation. 46SIS3 or DMSO was administered by i.p. injection four times a week for 4 weeks in 12-week-old VHL −KO ; Setd2 −KO mice.We found that the surface of SIS3-treated mice was relatively smoother compared to the control group (Figure 7A,B).BUN and creatinine levels were reduced in the blood of SIS3-treated mice compared to the control group (Figure 7C).In addition, the degree of kidney fibrosis was greatly reduced in SIS3-treated mice compared to the control group (Figure 7D,E).Meanwhile, p-Smad3, COL1A1 and α-SMA positive staining were reduced in drug-treated mice compared with controls, demonstrating that the TGF-β/Smad inhibitor can largely relieve the fibrotic phenotype (Figure 7F,G).The same results were observed in vitro (Figure 7H,I).Overall, these results suggested that inhibition of the TGF-β/Smad signalling pathway can rescue the renal fibrosis phenotype caused by SETD2 absence.

DISCUSSION
In this study, we demonstrated that SETD2 plays a critical role in the inhibition of the development of kidney fibrosis both in vitro and in vivo.We illustrated that SETD2 deficiency promotes renal fibrosis in VHL-deficient mice by the activation of the TGF-β/Smad signalling pathway.SETD2 maintains the expression level of Smad7 through H3K36me3 and inhibits the TGF-β/Smad signalling pathway.The anti-fibrotic effect of SETD2 provides an innovative insight into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.][25] SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes. 25Our recent studies reported that SETD2 plays important roles in intestinal immunity, 27 suppressing inflammatory bowel disease, 28 V(D)J recombination in normal lymphocyte development, 29 attenuating experimental colitis, 30 inhibiting pancreatic carcinogenesis, 31 bone marrow mesenchymal stem cell differentiation, 32 genomic imprinting, embryonic development, 33 spermiogenesis 34 and cutaneous wound healing. 35Based on the PKD model caused by the oncogene MYC, SETD2 deficiency accelerates the transition from PKD to RCC by regulating β-catenin activity at the transcriptional and post-transcriptional levels. 26In addition to the role of transcription regulation, SETD2 is also involved in non-histone methylation modifications, 47,48 alternative splicing, 49 etc.For example, it has been reported that SETD2 can restrict prostate cancer metastasis by integrating EZH2 and AMPK signalling pathways. 50Loss of SETD2 promotes acinar-to-ductal metaplasia and epithelial-mesenchymal transition during pancreatic carcinogenesis induced by Kras. 31here is evidence suggesting increased expression and activation of TGF-β/Smad in human kidney disease.2][53] TGFβ/Smad signalling pathway is the primary factor that drives renal fibrosis. 12Few studies have been conducted between SETD2 and the TGF-β/Smad signalling pathway, especially in the field of renal diseases.Smad7 is a negative feedback inhibitor of the TGF-β/Smad signalling pathway. 54mad7 can recruit Smurf1 and Smurf2 to degrade TGFR1.In addition, Smad7 can inhibit the TGF-β/Smad signalling pathway by competing with Smad2/3 for bounding to TGFR1 to prevent the phosphorylation of Smad2/3.SIS3 is a selective Smad3 inhibitor that attenuates TGF-β1dependent Smad3 phosphorylation and DNA binding. 46IS3 can alleviate many diseases, such as lung cancer. 55he results that SIS3 can treat fibrosis caused by SETD2 inactivation to some extent prove that the clinical application of SIS3 will be more desirable for patients with reduced SETD2 expression.
SETD2 and VHL are both commonly mutated genes in renal cell cancer.The expression of SETD2 and VHL is positively correlated in patients with renal fibrosis and renal cancer, indicating their clinical significance.Available studies have suggested that VHL protein can also ubiquitinate Smad3. 56,57In this study, we found that SETD2 and VHL could jointly inhibit TGF-β/Smad signalling pathway activation.Deletion of SETD2 results in reduced expression of Smad7, leading to the failure to inhibit the TGF-β signalling pathway.Meanwhile, the lack of VHL leads to the excessive accumulation of Smad3, which promotes the TGF-β signalling pathway.The combined effect of the two aspects results in the excessive activation of the TGFβ signalling pathway.However, as important regulatory molecules, SETD2 and VHL are involved in various biological processes.The potential interaction between VHL and SETD2 is unknown and undoubtedly complex.And it will also be a major focus of our future work.
In summary, we established a mouse model of renal fibrosis driven by deficiency of SETD2 and VHL.SETD2 deficiency resulted in severe renal fibrosis in the absence of VHL, and based on these results, we found that SETD2 deletion can lead to reduced expression of Smad7 by H3K36me3, which activates the TGF-β/Smad signalling pathway and leads to renal fibrosis.In addition, TGFβ inhibitor can rescue the phenotype caused by SETD2 absence.For clinical translation, pharmaceutical investigation of the cross-talk between SETD2 and TGF-β/Smad signalling may provide a potentially promising strategy to prevent renal fibrogenesis in patients (Supporting Information).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during this study are available from the corresponding author upon reasonable request.RNA sequencing raw data have been deposited in the Gene Expression Omnibus (GEO) under accession number GEO: GSE213922.ChIP sequencing: GSE125528.

R E F E R E N C E S
fl/fl mice were generated by Shanghai Biomodel Organism Co.The Ksp Cre mice (B6.Cg-Tg (Cdh16-cre) 91Igr/J) and VHL fl/fl were purchased from The Jackson Laboratory.Eight-week-old Setd2 fl/fl mice were bred with Ksp Cre mice, resulting in offspring with the genotype Ksp Cre ; Setd2 fl/fl mice (Setd2 −KO , n = 30).Eight-week-old VHL fl/fl mice were mated with Ksp Cre mice to generate Ksp Cre ; VHL fl/fl mice (VHL −KO , n = 30).Setd2 −KO mice were mated with VHL fl/fl mice to generate Ksp Cre ; VHL fl/fl Setd2 fl/fl mice (VHL −KO ; Setd2 −KO , n = 30).All mice used in this study were in C57 background and were maintained in a specific pathogen-free facility.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.The ethical number of animal experiments is 202201027.

F I G U R E 1
The mRNA expression of SET domain-containing 2 (SETD2) is positively correlated with Von Hippel-Lindau (VHL) in kidney.(A) The mRNA expression level of SETD2 in normal kidneys (n = 12) and transplant kidneys (n = 18, using dataset GSE135327).(B) The mRNA expression level of SETD2 in the kidneys with interstitial nephritis (ISN) (n = 5) and interstitial fibrosis/tubular atrophy (IFTA) (n = 5, using dataset GSE120495).(C) The double immunofluorescence (IF)staining experiment results of SETD2 with Lotus tetragonolobus lectin (LTL) and Tamm-Horsfall protein (THP).Scale bars: 50 μm.(D) The statistical results of the double IF staining experiments.(E) The mRNA expression level of VHL in normal kidneys (n = 12) and transplant kidneys (n = 18, using dataset GSE135327).(F) The relation of the expression of SETD2 and VHL in normal kidneys and transplant kidneys (n = 5, using dataset GSE135327).(G) The relation of expressions of SETD2 and VHL in the kidneys with ISN and IFTA (using dataset GSE120495).(H) The relation of expressions of SETD2 and VHL in normal and transplant kidneys from The Cancer Genome Atlas (TCGA).The data are represented as the mean ± standard deviation (SD).*p < .05,**p < .01,***p < .001,****p < .0001.